Influenza has a long history of pandemics, epidemics, resurgences and outbreaks. Avian influenza, including the H5N1 strain, is a highly contagious and potentially fatal pathogen, but it currently has only a limited ability to infect humans. However, avian flu viruses have historically observed to accumulate mutations that alter its host specificity and allow it to readily infect humans. In fact, two of the major flu pandemics of the last century originated from avian flu viruses that changed their genetic makeup to allow human infection. The emergence of new influenza A (H1N1) virus (henceforth: influenza A(H1N1)v virus, where v stands for variant, according to nomenclature agreed by the World Health Organization Global Influenza Surveillance Network—WHO GISN) in humans has led to the requirement for sensitive and specific assays for the differential diagnosis and confirmation of influenza A(H1N1)v virus infections, necessary to guide public health actions.
Triple-reassortant swine influenza viruses, which contain genes from human, swine, and avian influenza A viruses, have been identified in swine in the United States since 1998. In April, 2009, the U.S. Centers for Disease Control and Prevention identified novel swine-origin influenza A virus which contains a unique combination of gene segments from both North American and Eurasian swine lineages, colloquially called swine flu. Co-circulation of current seasonal human H1N1, H3N2, and Swine-Origin Influenza (S-OIV) A (H1N1) Viruses in the upcoming flu seasons poses a challenge for sub-typing individual strains and potential reassortants. The more complicated, and perhaps dangerous, scenario is the reassortment of Swine-Origin Influenza A with highly pathogenic avian H5N1 or with other serotypes.
Traditional methods for influenza detection and subtype identification are based on virus isolation in tissue culture. Methods for subtyping of the virus require expansion of viruses in embryonated eggs, followed by subtyping with serological methods (HI tests). This procedure might take a week and considerable effort. Although virus isolation has long served as the standard for the diagnosis of influenza virus, the approach alone is inefficient when worldwide outbreaks occur as it is less sensitive, time-consuming, and requires considerable technical expertise. The rapid shell vial culture assay, although more rapid than the standard tissue culture based method, the time to completion is still more than 24 h. Currently, more rapid methods with higher sensitivity and specificity have been developed to identify different subtypes of influenza virus for humans and for poultry; the serological method include immunodiffusion test, counter-Immune-electrophoresis, latex agglutination test and immunity fluorescence (IF) enzyme-linked immunosorbant assay (ELISA) and molecular biology methods include RT-PCR, nested RTPCR, rRT-PCR, nucleic acid sequence-based amplification, loop-mediated isothermal amplification, and DNA microarrays are not suitable for on-site use in field investigations or in clinical practice due to constraints such as test accessibility, and requirements for highly trained personnel, time-intensive procedures, appropriate containment laboratory facilities and elaborate instrumentation.
Limitations of most of the conventional diagnostic methods are lack of accuracy, sensitivity and delay in getting results. Importantly, the current standard for influenza sub-type identification relies on either a polymerase chain reaction test or culture approach, neither of which is a quick (or inexpensive) test. The current crop of quick test products on the market is only capable, due to limitations in their technology platform, of providing a yes/no answer to the presence of influenza. While swine-origin influenza virus (S-OIV; National Virus Reference Laboratory, NVRL, Dublin) real time PCR detection is available, it may not be applicable for more-complex reassortant strains. Available real-time PCR assays (HPA A(H1)v, CDC A (H1)v, HPA A(N1)v and NVRL S-OIV assays are suitable as first-line tests but accurate assessment requires concurrent use of primary diagnostic and confirmatory assays (Ellis J., et al. Euro Surveill. 2009; 14(22):pii=19230). Further, these methods require relatively sophisticated laboratories, which are sometimes unavailable or inconvenient for clinical application.
Glycans are typically the first and potentially the most important interface between cells and their environment. As vital constituents of all living systems, glycans are involved in recognition, adherence, motility and signaling processes. For example, all cells in living organisms, and viruses, are coated with diverse types of glycans; glycosylation is a form of post or co-translational modification occurring in all living organisms; and altered glycosylation is an indication of an early and possibly critical point in development of human pathologies. (Hirabayashi, J. 2003, Trends in Biotechnology 21 (4): 141-143; Hakomori, S-I. Tumor-associated carbohydrate antigens defining tumor malignancy: Basis for development of and cancer vaccines; in The Molecular Immunology of Complex Carbohydrates-2 (Albert M Wu, ed., Kluwer Academic/Plenum, 2001). These cell-identifying glycosylated molecules include glycoproteins and glycolipids and are specifically recognized by various glycan-recognition proteins. Carbohydrates are involved in inflammation, cell-cell interactions, signal transduction, fertility, bacteria-host interactions, viral entry, cell differentiation, cell adhesion, immune response, trafficking, and tumor cell metastasis.
Carbohydrates can also be expressed on the outer surface of a majority of viral, bacterial, protozoan, and fungal pathogens. The structural expression of carbohydrates can be pathogen-specific, making carbohydrates an important molecular target for pathogen recognition and/or infectious diseases diagnosis. Glycans, chains of sugars that often form complex branched structures on proteins or lipids, are major components of the outermost surface of many viruses. Receptor specificity for the influenza virus is usually controlled by the glycoprotein HA on the virus surface. Features of the differential binding among influenza virus suggest new flu as an intermediary genetic mixing vessel and facilitate a development of diagnostics. This pathogen specific expression of carbohydrates also can aid in vaccine development. Most interactions of virus with their hosts are influenced to an important degree by the pattern of glycans and glycan-binding receptors that each expresses. This holds true at all stages of infection, from initial colonization of host epithelial surfaces, to tissue spread, to the induction of host-cell injury are dominated by glycans (See FIG. 2). The two major surfaces proteins of the virus are hemagglutinin (HA) and neuraminidase (NA). The HA and NA are grouped into 16 and 9 subtypes, respectively, both have high sequence variability even within subtypes and thus provide an effective means of monitoring changes that might occur in a virus. The HA protein protrudes from the surface of the virus and allows it to attach to a cell to begin the infection cascade. The NA protein is also located on the surface of the virus and allows the release of new particles within the infected cell by cleaving the sialic acid moiety of cellular receptors.
The development of nucleotide and protein microarrays has revolutionized genomic, gene expression and proteomic research. ((Schena, M. et al. Science, 1995, 270:467-70; MacBeath, G. and Schreiber, S. L. 2000, Science, 289, 1760-1763). One feature of the post-genomic period is the exploration of biophysical, biochemical, and immunological properties of carbohydrate-carbohydrate and carbohydrate-protein interactions. Thus, a method is needed to study protein-carbohydrate interactions and to better understand these important biological processes.
Glycomics, the comprehensive study of glycomes, focuses on the interactions of carbohydrates with other biological processes. Carbohydrate microarrays are a platform for glycomic studies probing the interactions of carbohydrates with other biopolymers and biomaterials, in a versatile, rapid, and efficient manner. One particular advantage of the carbohydrate microarray is that a glycomic analysis requires only picomoles of a material and permits typically hundreds of interactions to be screened on a single microarray. The miniaturized array methodology is particularly well suited for investigations in the field of glycomics, since biological amplification strategies, such as the Polymerase Chain Reaction (PCR) or cloning, do not exist to produce usable quantities of complex oligosaccharides. Presenting carbohydrates in a microarray format can be an efficient way to monitor the multiple binding events of an analyte, such as, a protein interacting with one or more carbohydrates immobilized on a microarray surface.
The development of glycan microarrays has been very slow for a number of reasons. First, it is difficult to immobilize a library of chemically and structurally diverse glycans on arrays, beads or the like. Second, glycans are not readily amenable to analysis by many of the currently available molecular techniques (such as rapid sequencing and in vitro synthesis) that are routinely applied to nucleic acids and proteins. methods of preparing glycan arrays have been described in PCT/US2005/007370 filed Mar. 7, 2005 titled “High Throughput Glycan Microarrays” (Blixt), and U.S. Pat. App. Pub. No. 20080220988 (Zhou).
Microarray signals are detected by many technologies. Fluorescent labeling and detection is the most popular technique used to identify hybridization signals because it is sensitive and much easier and safer to handle than radioactive labeling methods (Parrish, M. L. et al. J. Neurosci. Methods, 2004, 132, 57-68). Sensitive fluorescence detection commonly uses a laser and a confocal microscope, e.g., DNA microarray detector made by Affymetrix Inc., which are typically very expensive and need a trained technician to operate.
The detection of protein analytes on microarrays has emerged as a powerful tool for proteomics as well as diagnostics (Macbeath, G. et al. Science (2000), 289, 1760-1763; Moody, M. D. et al. Biotechniques (2001), 31, 186-194; Nielsen, U. B. et al. Journal Immunol. Meth. (2004), 290, 107-120) A variety of different detection methods have been developed for labeling antibody arrays including, but not limited to, fluorescence, (Macbeath, G. et al. Science (2000), 289, 1760-1763; Li, Y. L. et al. (2003), 19, 1557-1566) chemiluminescence (Moody, M. D.; et al. Biotechniques 2001, 31, 186-194), resonance light scattering (Nielsen, U. B. et al. Journal Immunol. Meth. (2004), 290, 107-120), and SERS (Grubisha, D. S. et al. Anal. Chem. (2003), 75, 5936-5943). Signal amplification strategies such as rolling circle amplification (RCA) also have been used to increase the detection sensitivity of fluorescence-based strategies. (Schweitzer, B et al. Nat. Biotechnol. (2002), 20, 359-365; Wiltshire, S. et al. Proc. Natl. Acad. Sci. U.S.A. (2000), 97, 10113-10119) These methods have provided high sensitivity detection (<10 pg/mL) of protein analytes. However, the reproducible preparation of highly purified antibody reagents is both challenging and time consuming (Jayasena, S. D. Clin. Chem. (1999), 45, 1628-1650).
Rapid and simple diagnostic methods for confirming infection with influenza virus are urgently needed. Especially at the beginning of a new pandemic outbreak, an early differential diagnosis may alter clinical management, such as infection control procedures, consideration of antiviral treatment options and avoiding the inappropriate use of drugs. Thus. there is a need for novel techniques to classify subtypes of influenza viruses. There is a need for rapid and sensitive methods for detecting and classifying influenza viruses using glycan arrays.